Background/Aims: Acute kidney injury (AKI) is common, results in nonrenal sequelae, and predisposes patients to long-term cardiovascular disease. The long-term systemic effects of AKI remain unclear. Sex is an important biological variable in ischemia-reperfusion AKI, and the protective role of estrogen has stymied the inclusion of both sexes in preclinical AKI studies. ITF2357 is a nonspecific histone deacetylase inhibitor that has been shown to improve cardiac outcomes in murine models of hypertension. Here, we review recent work that provides new insight into our understanding of cardiovascular sequelae following AKI. Methods: Adult male and female C57BL/6J mice underwent 25 min (males) and 34 min (females) of bilateral ischemia-reperfusion AKI or sham procedure. A male treatment arm received chow containing the nonspecific histone deacetylase inhibitor ITF2357 starting 3 days after AKI. Serial renal function, echocardiograms, and blood pressure assessments were performed throughout the 1-year study; renal histology and cardiac and plasma metabolomics were evaluated at 1 year. Results: Measured glomerular filtration rates throughout the 1-year study showed that the female model of AKI matched the male model. Untreated males developed depressed diastolic function after AKI, whereas females and males treated with ITF2357 maintained normal diastolic function. Both untreated males and females developed hypertension after AKI; males treated with ITF2357 remained normotensive. Conclusions: Ischemic AKI results in long-term cardiovascular sequelae with sex as an important biological variable in outcomes. Histone deacetylase inhibition affects cardiovascular outcomes after AKI.

© 2022 S. Karger AG, Basel

Introduction

The short-term systemic sequelae of acute kidney injury (AKI) has been well-established, as has the risk for progression to chronic kidney disease. Growing epidemiological data demonstrate that a single episode of AKI significantly increases long-term cardiovascular morbidity and mortality [1]. In adult patients, AKI was found to be independently associated with cardiovascular events, especially heart failure, by 1 year after discharge [2]. While the cardiovascular complications of CKD in pediatric patients is well described [3], the impact of AKI on linear growth and cardiovascular health in pediatric patients is less well described. Little is known about the mechanisms that mediate the relationship between AKI and the long-term adverse events. Here, we review our recently published murine studies investigating the long-term systemic outcomes of bilateral ischemia-reperfusion AKI.

Studies of human and animal models of AKI indicate that AKI is a systemic disorder [4]. AKI results in a systemic inflammatory response which in turn affects distant organ function, and it is this distant organ function that persists after the AKI event [5]. It has been shown that vascular dysfunction is an independent predictor of CKD and future cardiovascular events in adults [6]. Prior murine models demonstrated mitochondrial injury and cardiac dysfunction after AKI; however, these studies were short term (<72 h) and included only males [7-9].

While the NIH has emphasized the importance of including male and female animals in preclinical research, most animal models of AKI use one sex or the other because of known sex differences in AKI outcomes. In models of ischemia-reperfusion injury, estrogen is known to protect against AKI, while testosterone is deleterious. For this reason, most preclinical ischemia-reperfusion models utilize males.

To clarify this issue, we performed a 1-year study evaluating the cardiorenal outcomes of AKI in male and female C57BL/6J wild-type mice. We performed bilateral ischemia-reperfusion injury or sham procedure on day 0 [10, 11]; 24 h later, we measured BUN and SCr. Animals in the AKI cohorts were excluded if the 24 h BUN was <70 and SCr <0.7 mg/dL. In order to overcome the protective effects of estrogen, females received a longer duration of ischemia time compared to males (34 min vs. 25 min). To assess renal function, the glomerular filtration rate was measured noninvasively serially throughout the year-long study. To assess cardiac outcomes, serial echocardiograms and blood pressure measurements were obtained. Animals were sacrificed 1 year after the AKI or sham procedure, and kidney fibrosis markers and cardiac and plasma metabolomics were assessed.

Matched Model of Ischemia-Reperfusion AKI between Males and Females

With the discordant ischemia duration between males and females, there was no difference between the measured GFR in males and females throughout the 1-year study, indicating a matched model of AKI severity between the sexes. At sacrifice 1 year after the procedure, serum creatinine and BUN were measured. There was no difference between SCr in females and males with AKI compared to sham controls. BUN, which is a more sensitive biomarker for kidney injury in our murine models, was persistently elevated at 1 year; however, there was no difference between male and female AKI cohorts [11].

Sex Biases in Cardiovascular Outcomes after AKI

After having determined that the model was matched with respect to the degree of AKI and longitudinal GFR, we next sought to determine whether there were changes with respect to sex on systemic outcomes. We performed echocardiograms at baseline, 6, 9, and 12 months. The E’ to A’ ratio is a measure of diastolic function. In healthy hearts, E’ should be greater than A’. Depression of the E’ to A’ ratio indicates diastolic dysfunction. Males with AKI had a depressed E’/A’ ratio at 6, 9, and 12 months, while females maintained normal diastolic function throughout the 1-year study (Fig. 1) [10, 11].

Fig. 1.

Diastolic function after AKI. Untreated males (AKI) had depressed diastolic function after AKI. Females (AKIF) and males treated with ITF2357 (AKIITF) maintained normal diastolic function after AKI. Adapted from Soranno et al. [10, 11].

Mean arterial blood pressure measurements were obtained via tail vein cuffs at 6, 9, and 12 months. Males with AKI were hypertensive at all study intervals. Females with AKI were hypertensive at 6 and 9 months but had normalized blood pressure by 12 months (Fig. 2) [10, 11]. Hypertension is a known cause of diastolic dysfunction. A 1 month, the male experiment was repeated to investigate whether hypertension preceded diastolic dysfunction. Instead, 1 month after AKI, males had diastolic dysfunction but not hypertension, implying that the diastolic dysfunction preceded elevation in blood pressure and therefore was not caused by hypertension alone [10].

Fig. 2.

Mean arterial blood pressure after AKI. Untreated males (AKI) and females (AKIF) developed hypertension after AKI. Males treated with ITF2357 (AKIITF) remained normotensive after AKI. Adapted from Soranno et al. [10, 11].

After sacrifice at 1 year, metabolomics analyses were performed on the cardiac tissue and plasma. In the plasma, numerous metabolites of interest indicated sex biases after AKI. In sum, the plasma findings indicated a difference in glutathione metabolism, which is related to reactive oxygen species. In the cardiac tissue and plasma, we found changes in arginine and nitrogen metabolism, which is important in reactive nitrogen species. Taken together, these data suggest changes in mitochondria-related oxidative stress in males and females.

The Effect of Histone Deacetylase Inhibition on Cardiorenal Outcomes

Histone deacetylase inhibitors regulate epigenetic gene expression and are used clinically in both oncologic and psychiatric diseases. ITF2357 is a nonspecific histone deacetylase inhibitor that has been shown to improve cardiac outcomes in murine models of cardiac dysfunction [12, 13]. In the male cohorts, we included a study arm of AKI and sham cohorts treated with ITF2357 containing chow. We started the chow 3 days after AKI or sham procedure, to mimic a clinically feasible intervention. Males with AKI that were treated with ITF2357 had the same measured GFR as untreated AKI cohorts but did not develop either diastolic dysfunction (Fig. 1) or hypertension (Fig. 2). They did however have increased kidney fibrosis at 1 year. Cardiac metabolomic analysis comparing the male cohorts untreated versus treated with ITF2357 demonstrated improvement in cardiac energy metabolism in the ITF2357 cohorts.

The Role of Cardiac ATP

Given the metabolomics data in both the female versus male and treated versus untreated male cohorts suggesting differences in cardiac energy metabolism, we next measured levels of ATP in the cardiac tissue from the 12-month samples. Untreated males with AKI had reduced levels of cardiac ATP. However, both females and males treated with ITF2357 had normal levels of cardiac ATP [10, 11]. These findings correlated with the diastolic function findings. Reduced levels of cardiac ATP are a known cause of diastolic impairment.

Kidney Fibrosis Outcomes

Finally, to complete our cardiorenal assessment, we measured kidney fibrosis markers. Upon quantification of Picrosirius red, collagen 3 immunohistochemistry, and hydroxyproline, both females and ITF2357 treated males with AKI demonstrated even more kidney fibrosis than the untreated AKI males [10, 11].

The Impact of AKI on Growth Outcomes

One final long-term systemic finding in both sexes was that growth was impaired after AKI. In the male study, males with AKI demonstrated significantly decreased weight compared to shams starting at 3 months and cohorts treated with ITF2357 had even less weight gain [10]. The ITF2357 cohorts also demonstrated a reduction in muscle mass, whereas the untreated males did not. In females, AKI resulted in reduced growth at 1 year but did not result in any muscle loss compared to shams [11].

Conclusions and Future Directions

In conclusion, we have developed a matched model of AKI in male and female mice out to 1 year. Males developed diastolic dysfunction followed by hypertension. Clinically, it is often assumed that diastolic dysfunction is a result of preceding hypertension. Our findings suggest that other causes, such as reduced cardiac ATP stores, may be worth investigating. Female sex protects against diastolic dysfunction but not hypertension. In males, ITF2357 protects against both diastolic dysfunction and hypertension. Changes in energy metabolism may explain the differential outcomes. Despite cardiovascular protection, females had worse renal fibrosis, correlating with increased ischemia time. ITF2357 did not prevent renal fibrosis or impact measured GFR but did impact both weight and muscle mass. AKI resulted in decreased weight in both males and females.

To date, the long-term systemic sequelae of AKI remain unknown in patients. Future investigations are needed to determine whether these findings translate into humans and whether sex biases are identified. In light of the long-term growth findings in the murine studies, the effect of severe AKI on childhood growth warrants investigation.

Acknowledgments

The authors would like to acknowledge the Ludeman Family Center for Women’s health Research for research training in sex as a biological variable.

Statement of Ethics

The animal studies reviewed herein were approved by the University of Colorado’s Animal Care and Use Committee and adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals [10, 11].

Conflict of Interest Statement

The authors report no relevant conflicts of interests. Dr. Gist is a consultant for Bioporto and received honoraria from Medtronic in 2021.

Funding Sources

Funding for this study was provided by the Consortium for Fibrosis Research & Translation (CFReT), supported through the University of Colorado School of Medicine’s Transformational Research Funding initiative, and the NIDDK career development award program (DES, K08 DK109226-01A1).

Author Contributions

Danielle E. Soranno and Katja M. Gist contributed to the writing of the manuscript. Danielle E. Soranno compiled the figures, adapted from previously published work cited herein.

Data Availability Statement

Primary data will be made available upon reasonable request to the corresponding author.

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